Covalent attachment of the Small ubiquitin related modifier, SUMO, to proteins important for regulated gene expression has been correlated with inhibition of transcription. Post-translational modification by SUMO represses activity of the dual function transcription factor Sp3. Sp3 is required for post-natal survival and differentiation of bone, tooth, and hematopoietic lineages in mice. Polymorphisms in binding sites for Sp3 and the related Sp1 protein correlate with disease risk for osteoporosis, diabetes, and cancer. The studies in this proposal investigate the hypothesis that SUMO-modified Sp3 promotes formation of a repressive chromatin structure by recruiting a corepressor complex containing a SUMO-binding domain. Non-covalent interactions with SUMO-modified transcription factors is proposed to contribute to gene targeting by several chromatin modifying complexes. The specific aims of this research are to (1) investigate how SUMO modification regulates protein interactions with Sp3, (2) analyze the molecular mechanisms of repression by SUMOylated Sp3 in vivo, and (3) determine the identity and function of SUMO binding domains in chromatin modifying complexes that interact non-covalently with SUMO. These studies will identify cofactors and mechanisms underlying Sp3-dependent regulation of genes important for cell proliferation, differentiation, and survival. These studies will provide new insights into the molecular mechanisms by which SUMO modification of many transcription factors inhibits transcription, determine the structure and function of SUMO binding domains in corepressors, and provide a foundation to determine the molecular basis for context-dependent activities of Sp3 and SUMO. Studies of non-covalent SUMO binding by chromatin modifying complexes will increase understanding of the epigenetic mechanisms that regulate chromatin structure and gene expression in normal development and disease. Relevance: These studies address the fundamental mechanisms by which the SUMO protein regulates gene expression in human cells. Aberrant patterns of SUMO attachment to other proteins has been associated with pathogenic viral and bacterial infections, diabetes, cancer, and neurodegenerative diseases. Therefore, increased understanding of the fundamental mechanisms underlying SUMO function in regulation of gene expression may lay the foundation for novel therapeutic strategies that address these significant human health issues.